Elevated levels of apoB-100 containing lipoproteins and markers of systemic inflammation are often observed in patients with cardiovascular diseases. The concentrations can be reduced by pharmacotherapy or extracorporeal treatment. The sorbent, which removes CRP and atherogenic lipoproteins, simultaneously reduces the bloodstream concentration of these components. The efficacy and selectivity of the designed sorbent were studied, desorption constants of CRP (Kd = 4.2 × 10-8 M) and LDL (Kd = 7.7 × 10-7 M) were distribution coefficients of CRP (Kc = 101) and Lp(a) (Kc = 38) were calculated, and the ability to bind large amounts of atherogenic lipoproteins (up to 32 mg of TC per mL of the sorbent gel) was demonstrated. Our sorbent can be recommended for performing complex removal of CRP and atherogenic lipoproteins from the blood plasma in patients with refractory hyperlipidemia and CVD that are accompanied by elevated levels of CRP.

— Different methods of covalent lysozyme immobilization have been compared to choose an optimal approach to the development of a material suitable for medical applications in extracorporeal therapy. A novel method for lysozyme immobilization on a polymeric agarose matrix has been proposed, which provides the effective action of lysozyme on bacterial cells and eliminates the leakage of the enzyme from the material. The resultant immobilized lysozyme exhibits bacteriolytic activity toward Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Escherichia coli . During the immobilization of the enzyme, a broadening of the pH optimum of its activity occurs. The compatibility of immobilized lysozyme with human whole blood has been shown.

•Chemical modification with benzaldehyde and anisaldehyde affects only 1–2 amino groups of lysozyme molecule.•Chemical modification does not prevent further covalent immobilization of the protein using the remaining free amino groups.•Composite material based on modified covalently immobilized lysozyme shows excellent endotoxin adsorption.•The adsorption of IgG on immobilized lysozyme did not increase after chemical modification.•The ability to specifically bind to IgG may be associated with the opsonin function of lysozyme.

The emergence of new antibiotic‐resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram‐positive and Gram‐negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram‐negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram‐positive Micrococcus luteus. Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus. When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 108 colony‐forming units·mL−1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram‐positive and Gram‐negative bacteria. The Michaelis constant (Km) values of immobilized lysozyme were increased by 1.5‐fold for E. coli cell lysis and 4.6‐fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram‐negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.

It was demonstrated for the first time that immobilized lysozyme can efficiently remove Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (endotoxins) from solutions. Experimentally confirmed sorption capacity for the developed sorbent was at least 400 ng of endotoxin per ml sorbent. The new sorbent is compatible with the whole human blood and can be potentially used in extracorporeal therapy in the treatment of sepsis.

Цель исследования. Методы терапевтического афереза в мировой клинической практике считаются эффективными для лечения тяже5 лых заболеваний аутоиммунной природы в качестве как первой, так и второй линии терапии. В настоящее время для специфического удаления иммуноглобулинов (Ig) широко используются различные плазмосорбенты с иммобилизованными природными и синтетически5 ми лигандами. Нами был синтезирован новый сорбент, пригодный для гемоперфузии. Предлагаемая работа посвящена исследованию свойств гемосорбента в сравнении с одним из широко используемых плазмосорбентов. Материал и методы. В работе были использованы сорбент с поликлональными антителами к иммуноглобулинам G человека (плазмосорбент) и сорбент с синтетическим лигандом, иммо5 билизованным на макрогранулированную матрицу (гемосорбент). Оценивали динамику концентрации различных классов и подклассов IgG, специфических антифосфолипидных и анти5ДНК5 аутоантител, а также С5реактивного белка (СРБ) в условиях проведения экспе5 риментов in vitro. Результаты. Исследованные сорбенты обладали сравнимой эффективностью удаления IgG и IgА (60 и 32 против 47 и 35 на плазмо5 и гемосорбенте соответственно). Однако IgM связывались только с плазмосорбентом (28). При этом гемосорбент с большей эффективностью связывал подклассы IgG3 и IgG4, а также специфические аутоантитела против двухцепочечной (дц) и денату5 рированной одноцепочечной (оц) ДНК. В отличие от плазмосорбента гемосорбент был способен с высокой эффективностью связывать и удалять из плазмы СРБ. Гемосорбент с иммобилизованным синтетическим лигандом был инертен относительно клеток крови и обладал гемосовместимостью в экспериментах in vitro при перфузии цельной крови. Выводы. Как плазмосорбент, так и гемосорбент, одинаково эффективно удаляют Ig основных классов (IgG и IgA), подклассы IgG и специфические антифосфолипидные и анти5ДНК аутоантитела. Создание сорбента для удаления IgG, подходящего для перфузии цельной крови, позволит расширить применение методов терапевтиче5 ского афереза для лечения пациентов с аутоиммунными и, возможно, сопутствующими сердечно5сосудистыми заболеваниями.Background. Therapeutic apheresis methods are applied in the world clinical practice as one of the effective approaches for treatment of severe autoimmune diseases, both as first5line and second5line therapy. Currently plasmаsorbents with immobilized native and synthetic ligands are widely used for specific removal of immunoglobulins. We have developed and investigated the new hemosorbent and compared it with one of the used plasmasorbents. Methods. In our study weve compared immunosorbent with sheep polyclonal antibodies against human immunoglobulin G and the sorbent with synthetic ligand, immobilized on cross5linked macro beads agarose matrix. Weve tested in vitro at the same conditions the efficiency of removal of Ig classes and subclasses, autoantibodies against DNA and antiphospholipid autoantibodies, and also C5reactive protein. Results. Both sorbents have almost equal efficiency of IgG and IgA removal 60 and 32 for plasmasorbent and 47 and 35 for hemosorbent. But IgM were removed only by plasmasorbent (28). The hemosorbent have binded more effective such subclasses of immunoglobulins as IgG3 and IgG4, and also specific autoantibodies to single or double stranded DNA. Hemosorbent have removed C5reactive protein very effective. Hemocompatibility of hemosorbent was verified by inertness with human blood cells. Conclusions. Ig5plasmasorbent and Ig5hemosorbent have the same efficacy for removal of Ig classes, IgG subclasses, antiphospholipid and others pathogenic autoantibodies. But Ig5hemosorbent is more successful in C5reactive protein binding compared to Ig5plasmasorbent and it is hemocompatible.

The data of the turbidimetric measurement for the enzymatic lysis of various living bacterial cells are analyzed. A method for the correct recalculation of the turbidimetric data (–Δ A /Δ t ) into absolute values of the change in the concentration of living cells (–ΔCFU/Δ t ) is proposed. The dimensionless efficiency of cell lysis–(1/CFU 0 ) · ΔCFU/Δ t for various bacterial cells is calculated to correctly compare the efficiency of the action of different bacteriolytic factors on various bacterial cells.

The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various substances has been studied. Glycine and lysine do not affect the activity of interleukin-2 but increase that of lysozyme, showing a bell-shape concentration dependence peaking at 1.5 mM glycine and 18 mM lysine. Arginine and glutamate activate both interleukin-2 and lysozyme with a concentration dependence of the saturation type. Aromatic amino acids have almost no effect on the activity of both interleukin-2 and lysozyme. Aromatic amines, tryptamine, and tyramine activate interleukin-2 but inhibit lysozyme. Peptide antibiotics affect interleukin and lysozyme similarly and exhibit maximum activity in the micromolar range of antibiotics. Taurine has no effect on the activity of interleukin-2 and lysozyme. Mildronate showed no influence on lysozyme, but it activated interleukin-2 with the activity maximum at 3 mM. EDTA activates both interleukin-2 and lysozyme at concentrations above 0.15 mM.

The bacteriolytic activity of interleukin-2 and hen egg white lysozyme against 34 different species of microorganisms has been studied. It was found that 6 species of microorganisms are lysed in the presence of interleukin-2. All interleukin-2-sensitive microorganisms belong either to the Enterobacteriaceae, Bacillaceae, or the Lactobacillaceae family. It was also found that 12 species of microorganisms are lysed in the presence of lysozyme, and 16 species of microorganisms are lysed in the presence of sodium dodecyl sulfate (SDS). The bacteriolytic activity of interleukin-2 and lysozyme was studied at various pH values.

Предложена новая методика получения аффинных сорбентов на основе лигандов – тирамина и триптамина. Для полученных хроматографических материалов были исследованы сорбционные характеристики. Показано, что триптамин-сефароза и тирамин-сефароза эффективно связывают IgG, IgA, липопротеид (а) (Lp(a)) и липопротеиды низкой плотности (LDL) из плазмы крови. Сорбционная емкость (мг/мл геля) составила IgG – 4–9, IgA – 2–4, Lp(a) – 3–5 и LDL – 5–7. Обнаружено, что Lp(a) и IgG могут связываться с сорбентом как независимо, так и в виде комплекса Lp(a) с IgG, существование которого может говорить о наличии у некоторых пациентов аутоантител к Lp(a). Новые сорбенты просты в синтезе, стабильны при использовании и хранении, могут применяться в медицинских и биотехнологических целях для связывания Lp(a), LDL, IgG, IgA.

A new procedure for the preparation of affinity sorbents based on tyramine and tryptamine ligands has been proposed. Sorption characteristics for the developed chromatographic materials were investigated. It was shown that tryptamine-sepharose and tyramine-sepharose efficiently bind IgG, IgA, lipoprotein (a) (Lp(a)) and low density lipoproteins (LDL) from blood plasma. The sorption capacity was 4–9 mg of IgG, 2–4 mg of IgA, 3–5 mg of Lp(a), and 5–7 mg of LDL per mL of gel. It was found that Lp(a) and IgG can bind to the sorbent either as themselves or as a complex of Lp(a) with IgG, the existence of which may indicate the presence of autoantibodies to Lp(a) in some patients. New sorbents are easy in synthesis, stable during use and storage, and they can be used for medical and biotechnological purposes to bind Lp(a), LDL, IgG, and IgA.

Получены гемосовместимые аффинные сорбенты на основе полисахаридной матрицы и лигандов – пептидов WY, WTY, WNY, связывающиe иммуноглобулины G человека (IgG). Проведено сравнение характеристик сорбентов по связыванию как общего IgG так и подклассов IgG. Обнаружено, что все новые сорбенты имеют хорошие характеристики по удалению IgG из плазмы крови, но сорбент на основе WNY в полтора раза эффективней остальных связывает IgG подкласса 3 (IgG3). Определены физико-химические характеристики процесса связывания IgG. Для сорбентов на основе пептидов WY, WTY, WNY константы десорбции IgG составили 10 ± 3, 28 ± 4 и 13 ± 3 мкМ. Максимальная расчетная сорбционная емкость по IgG составила 43 ± 2, 45 ± 3 и 46 ± 3 мг IgG на 1 мл сорбента. Показана гемосовместимость сорбентов, пригодность для медицинского использования.

The article continues studies of the recently discovered bacteriolytic activity of interleukin-2. It was detected earlier that interleukin (IL-2) possesses greater substrate specificity in comparison with chicken egg lysozyme. IL-2 disrupted the cell wall of Escherichia coli but did not lyse lysozyme substrates such as the cell walls of Micrococcus luteus and Bacillus subtilis . In the present study it is demonstrated for the first time that both IL-2 and chicken egg lysozyme are capable of lysing Lactobacillus plantarum . The effects of IL-2 and chicken egg lysozyme on Lactobacillus plantarum are compared with those on Escherichia coli . The dependences of the rate of lysis on the concentration of bacteriolytic factors and pH are studied.